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. 2020 Nov 6;218(2):e20192203. doi: 10.1084/jem.20192203

Figure 2.

Figure 2.

Concentration of retrovirus and optimal T cell activation increases transduction efficiency. (A) Diagram of the 2G VEGFR-2–targeted CAR construct (DC101-28z) comprising an Igκ chain leader sequence, the hinge and transmembrane (TM) region of CD8α, the costimulatory ED of CD28, and the ED of CD3ζ (DC101, αVEGFR-2 scFv; L, linker; VH, variable heavy chain; VL, variable light chain). (B) Comparison of activation state of differently stimulated T cells. Numbers represent mean percentage of expression ± SEM of T cells from n = 6 mice pooled from two independent experiments. (C) Virus titer with and without concentration by ultracentrifugation as determined with transduced T cells on day 7 after transduction. Numbers represent mean MOI ± SEM calculated in transduced T cells from n = 3 mice. TU, transducing units. (D) CAR expression on T cells transduced with serially diluted retrovirus as evaluated by flow cytometric analysis. Numbers represent mean percentage of CAR expression ± SEM of T cells from n = 3 mice. Representative histograms of transduced T cells with the indicated dilutions of retroviral supernatants shown. (E) Murine T lymphocytes were transduced once (1×) or twice (2×) after activation (24 and/or 48 h) at increasing MOI. Numbers represent mean percentage of MFI CAR ± SEM of T cells from n = 3 mice. (F) Representative histograms showing CAR frequency and expression level at day 7 after transduction. The experiments in C–Fwere repeated three times. (G) Schematic diagram of different murine T cell activation and transduction methods tested. (H) Evaluation of CAR frequency following different activation methods. Numbers represent mean percentage of CAR ± SEM of T cells from n = 5 mice pooled from two independent experiments. (I) Representative histograms of CAR expression for differently activated T cells at day 7 after transduction. Statistical analyses were performed using a one-way ANOVA with Tukey post hoc correction test (B and H) and a two-tailed unpaired Student’s t test (C): *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.